Two-Chamber Dual-Pore Device

ABSTRACT

Provided is a device comprising a channel through and defined by a plurality of layers surrounding the channel, the channel connecting a first and a second chambers separated by the plurality of layers, wherein the plurality of layers comprise a first layer, a second layer; and a conductive middle layer disposed between the first and second layers, wherein the channel comprises (a) a first region defined by the first layer, denoted as an inlet, that is about 0.5 nm to about 100 nm in diameter and (b) a second region defined by the second layer, denoted as an outlet, wherein the inlet and the outlet are about 10 nm to about 1000 nm apart from each other, and wherein the first and second chambers and the middle layer are connected to a power supply. Also provided are methods of preparing and using the device, in particular for nucleic acid sequencing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/358,001, filed May 13, 2014, which claims the benefit under 35 U.S.C.§ 371 of International Patent Application No. PCT/US2012/064879, filedNov. 13, 2012, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 61/629,184 filed Nov. 14, 2011,entitled “Microfluidic Chip Implementation of Dual-Nanopore ElectronicsConfiguration to Co-Trap Individual DNA Molecules and Methods of Use”,the contents of which are incorporated by reference in their entiretyinto the present disclosure.

BACKGROUND

A nanopore is a nano-scale conduit that forms naturally as a proteinchannel in a lipid membrane (a biological pore), or is engineered bydrilling or etching the opening in a solid-state substrate (asolid-state pore). When such a nanopore is incorporated into ananodevice comprising two chambers which are separated by the nanopore,a sensitive patch-clamp amplifier can be used to apply a trans-membranevoltage and measure ionic current through the pore.

Nanopores offer great promise for inexpensive whole genome DNAsequencing. In this respect, individual DNA molecules can be capturedand driven through the pore by electrophoresis, with each capture eventdetected as a temporary shift in the ionic current. The sequence of aDNA molecule can then be inferred from patterns within the shifted ioniccurrent record, or from some other auxiliary sensor in or near thenanopore, as DNA passes through the pore channel.

In principle, a nanopore sequencer can eliminate the needs for sampleamplification, the use of enzymes and reagents used for catalyticfunction during the sequencing operation, and optics for detection ofsequencing progress, some or all of which are required by theconventional sequencing-by-synthesis methods.

Electric nanopore sensors can be used to detect DNA inconcentrations/volumes that are no greater than what is available from ablood or saliva sample. Additionally, nanopores promise to dramaticallyincrease the read-length of sequenced DNA, from 450 bases to greaterthan 10,000 bases.

There are two principle obstacles to nanopore sequencing: (1) the lackof sensitivity sufficient to accurately determine the identity of eachnucleotide in a nucleic acid for de novo sequencing (the lack ofsingle-nucleotide sensitivity), and (2) the ability to regulate thedelivery rate of each nucleotide unit through the nanopore duringsensing. These two obstacles are often inter-related as the inability toregulate delivery rates is one of the underlying problems can beassociated with the lack of single-nucleotide sensitivity. Statedanother way, if the DNA is traversing past the sensor too rapidly, thenthe sensor's function can be compromised. While many research groups aredeveloping and improving nanopores to address obstacle 1, there is nomethod for addressing obstacle 2 that does not involve the use ofenzymes or optics, both of which work only in specialized nanoporetechniques and which incur higher complexity and cost compared to purelyelectrical methods.

SUMMARY

In one embodiment, the present disclosure provides a device, the devicecomprising a channel through and defined by a plurality of layerssurrounding the channel, the channel connecting a first and a secondchambers separated by the plurality of layers, wherein the plurality oflayers comprise: a first layer; a second layer; and a conductive middlelayer disposed between the first and second layers, wherein: the channelcomprises (a) a first region defined by the first layer, denoted as aninlet, that is about 0.5 nm to about 100 nm in diameter and (b) a secondregion defined by the second layer, denoted as an outlet, wherein theinlet and the outlet are about 10 nm to about 1000 nm apart from eachother, and wherein the first and second chambers and the middle layerare connected to a power supply.

In one embodiment, the inlet has a depth of from about 0.1 to about 100nm. In one aspect, wherein the outlet has a diameter greater than thediameter of the inlet. In one aspect, the inlet is about 1 nm to about20 nm in diameter. In another aspect, the inlet is about 0.2 nm to about10 nm in depth. In one embodiment, the inlet and outlet aresubstantially coaxial.

In some aspects, the first layer and/or the second layer comprises adielectric material. In one aspect, the first layer and/or the secondlayer comprises a metallic material and wherein the device furthercomprises insulating material between the first layer and the middlelayer and/or between the middle layer and the second layer. In someaspects, the first layer and/or the second layer comprises a materialselected from the group consisting of silicon, silicon nitride, silicondioxide, graphene, carbon nanotubes, TiO₂, HfO₂, Al₂O₃, metallic layers,glass, biological nanopores, membranes with biological pore inserts, andcombinations thereof.

In one embodiment, the middle layer comprises a metal selected fromchrome, aluminum, platinum, and gold. In another alternative embodiment,the middle layer comprises a metal that can be deposited with thin filmmethods such as e-beam evaporation, thermal evaporation, molecular beamepitaxi (MBE), or atomic layer deposition (ALD).

The device, in one embodiment, can further comprise the power supplywherein the power supply is configured to provide a first voltagebetween the first chamber and the middle layer, and a second voltagebetween the middle layer and the second chamber, and wherein the firstvoltage and the second voltage are independently adjustable. In oneaspect, the power supply comprises a voltage-clamp system or apatch-clamp system to generate each of the first and second voltages. Inone aspect, the middle layer is adjusted to be ground relative to thetwo voltages.

In some aspects, the device further comprises at least one sensorcapable of identifying a polymer or individual components of a polymerduring movement of the polymer through the inlet and outlet. In oneaspect, the sensor measures an ionic current across the inlet.

In one aspect, the sensor is configured to identify the polymer or theindividual components by measuring a current, a voltage, pH, an opticalfeature, or residence time associated with the polymer or one or morecomponents of the polymer.

In one aspect, the sensor is configured to form a tunnel gap allowingthe polymer to pass through the tunnel gap when the polymer is loadedwithin the inlet and outlet.

It is contemplated that the plurality of layers can be continuousregions of an integrated piece, or alternatively the plurality of layersare discontinuous.

Also provided, in one embodiment, is a method for controlling themovement of a charged polymer through a pore, comprising: (a) loading asample comprising a charged polymer in one of the first or secondchamber of the device, wherein the device is connected to avoltage-clamp or patch-clamp system for providing a first voltagebetween the first chamber and the middle layer, and a second voltagebetween the middle layer and the second chamber; (b) setting an initialfirst voltage and an initial second voltage so that the polymer movesthrough the chambers, thereby locating the polymer across both the inletand outlet; and (c) adjusting the first voltage and the second voltageso that both voltages generate force to pull the charged polymer awayfrom the middle layer, wherein the two voltages are different inmagnitude, under controlled conditions, so that the charged polymermoves through the channel and both the inlet and the outlet in onedirection and in a controlled manner.

In one aspect, the charged polymer is a polynucleotide. In anotheralternative aspect the charged polymer is a polypeptide. Other chargedpolymers provided by the invention include phospholipids,polysaccharides, and polyketides

In one aspect, the adjusted first voltage and second voltage at step (c)are about 10 times to about 10,000 times as high, in magnitude, as thedifference between the two voltages.

Still further provided, in one aspect, is a method for preparing adevice of the present disclosure, comprising drilling a channel througha plurality of layers comprising a first layer, a second layer and aconductive middle layer disposed between and insulated from the firstand second layers. In some aspects, the method further includesshrinking one or more of the plurality of layers to reduce the diameterand/or depth of the inlet and/or the outlet. In some aspects, thedrilling is carried out by focused ion beam.

In another exemplary embodiment, the device comprises a nanopore inputhead, comprising: a first nanopore having a first pore opening andenclosing a first ionic conductor; a second nanopore having a secondpore opening and enclosing a second ionic conductor; a nanopore chamberbetween said first nanopore and said second nanopore, said nanoporechamber enclosing an chamber ionic conductor that is biasedsubstantially at electrical ground, said nanopore chamber having bodyhaving a first aperture aligned with said first pore opening and asecond aperture aligned with said second pore opening; a firstdifferential amplifier having a first non-inverting input operativelyconnected to said first ionic conductor, a first inverting input, and afirst output; and a second differential amplifier having a secondnon-inverting input operatively connected to said second ionicconductor, a second inverting input, and a second output; wherein awidth of said nanopore chamber is less than 1 micron (μm).

The invention also discloses a method of capturing a strand of DNAcomprising the steps of: disposing first and second nanopore sensorswithin 1 micron of each other and within an ionic chamber; electricallygrounding said ionic chamber to electrically isolate the first andsecond nanopore sensors; applying a first voltage to the first nanoporesensor to capture part of a strand of DNA; and applying a second voltageto the second nanopore sensor to capture another part of the strand ofDNA.

The invention further discloses a nanopore system, comprising thesolid-state nanopore systems and associated circuits disclosed here.

The nanopore device systems may comprise cis and trans chambersconnected by an electrical communication means. In one embodiment thechambers comprise a medium, the medium selected from the groupconsisting of an aqueous medium, a non-aqueous medium, an organicmedium, or the like. In one embodiment the medium is a fluid. In analternative embodiment the medium is a gas. In one embodiment theelectrical communication means is a solid state pore comprising, forexample, silicon nitride, bifunctional alkyl sulfide, and/or gold orother metal or alloy. In the alternative, the cis and trans chambers areseparated by a thin film comprising at least one pore or channel. In onepreferred embodiment, the thin film comprises a compound having ahydrophobic domain and a hydrophilic domain. In a more preferredembodiment, the thin film comprises a phospholipid. The devices furthercomprise a means for applying an electric field between the cis and thetrans chambers. In one embodiment the pore or channel accommodates apart of the polyion. In another embodiment the pore or channelaccommodates a part of the molecule. In one preferred embodiment, themolecule is a macromolecule. In another preferred embodiment the polyionis selected from the group consisting of polynucleotides, polypeptides,phospholipids, polysaccharides, and polyketides.

In one embodiment the compound comprises enzyme activity. The enzymeactivity can be, for example, but not limited to, enzyme activity ofproteases, kinases, phosphatases, hydrolases, oxidoreductases,isomerases, transferases, methylases, acetylases, ligases, lyases,ribozyme, and the like. In a more preferred embodiment the enzymeactivity can be enzyme activity of DNA polymerase, RNA polymerase,endonuclease, exonuclease, DNA ligase, DNase, uracil-DNA glycosidase,kinase, phosphatase, methylase, acetylase, glucose oxidase, ribozyme,and the like.

In another embodiment the pore is sized and shaped to allow passage ofan activator, wherein the activator is selected from the groupconsisting of ATP, NAD⁺, NADP⁺, diacylglycerol, phosphatidylserine,eicosinoids, retinoic acid, calciferol, ascorbic acid, neuropeptides,enkephalins, endorphins, 4-aminobutyrate (GABA), 5-hydroxytryptamine(5-HT), catecholamines, acetyl CoA, S-adenosylmethionine, hexose sugars,pentose sugars, phospholipids, lipids, glycosyl phosphatidyl inositols(GPIs), and any other biological activator.

In another embodiment the pore is sized and shaped to allow passage of amonomer, wherein the monomer is selected from the group consisting ofdATP, dGTP, dCTP, dTTP, UTP, alanine, cysteine, aspartic acid, glutamicacid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagines, proline, glutamine, arginine, serine,threonine, valine, tryptophan, tyrosine, hexose sugars, pentose sugars,phospholipids, lipds, and any other biological monomer.

In yet another embodiment the pore is sized and shaped to allow passageof a cofactor, wherein the cofactor is selected from the groupconsisting of Mg²⁺, Mn²⁺, Ca²⁺, ATP, NAD⁺, NADP⁺, and any otherbiological cofactor.

In one preferred embodiment, the dual-nanopore system comprises asolid-state nanopore comprising, for example, a layer of graphene orother similar composition.

In an alternative embodiment the pore or channel comprises a biologicalmolecule, or a synthetic modified or altered biological molecule. Suchbiological molecules are, for example, but not limited to, an ionchannel, such as α-hemolysin, a nucleoside channel, a peptide channel, asugar transporter, a synaptic channel, a transmembrane receptor, such asGPCRs, a receptor tyrosine kinase, and the like, a T-cell receptor, anMHC receptor, a nuclear receptor, such as a steroid hormone receptor, anuclear pore, or the like.

In an alternative, the compound comprises non-enzyme biologicalactivity. The compound having non-enzyme biological activity can be, forexample, but not limited to, proteins, peptides, antibodies, antigens,nucleic acids, peptide nucleic acids (PNAs), locked nucleic acids(LNAs), morpholinos, sugars, lipids, glycosyl phosphatidyl inositols,glycophosphoinositols, lipopolysaccharides, or the like. The compoundcan have antigenic activity. The compound can have ribozyme activity.The compound can have selective binding properties whereby the polymerbinds to the compound under a particular controlled environmentalcondition, but not when the environmental conditions are changed. Suchconditions can be, for example, but not limited to, change in [H⁺],change in environmental temperature, change in stringency, change inhydrophobicity, change in hydrophilicity, or the like.

In one embodiment the macromolecule comprises enzyme activity. Theenzyme activity can be, for example, but not limited to, enzyme activityof proteases, kinases, phosphatases, hydrolases, oxidoreductases,isomerases, transferases, methylases, acetylases, ligases, lyases, andthe like. In a more preferred embodiment the enzyme activity can beenzyme activity of DNA polymerase, RNA polymerase, endonuclease,exonuclease, DNA ligase, DNase, uracil-DNA glycosidase, kinase,phosphatase, methylase, acetylase, glucose oxidase, or the like. In analternative embodiment, the macromolecule can comprise more that oneenzyme activity, for example, the enzyme activity of a cytochrome P450enzyme. In another alternative embodiment, the macromolecule cancomprise more than one type of enzyme activity, for example, mammalianfatty acid synthase. In another embodiment the macromolecule comprisesribozyme activity.

In an alternative, the macromolecule comprises non-enzyme biologicalactivity. The macromolecule having non-enzyme biological activity canbe, for example, but not limited to, proteins, peptides, antibodies,antigens, nucleic acids, peptide nucleic acids (PNAs), locked nucleicacids (LNAs), morpholinos, sugars, phospholipids, lipids, glycosylphosphatidyl inositols, glycophosphoinositols, lipopolysaccharides, orthe like. The macromolecule can have polynucleotide-binding activityand/or polypeptide biosynthesis activity, such as, but not limited to, aribosome or a nucleosome. The macromolecule can have antigenic activity.The macromolecule can have selective binding properties whereby thepolymer binds to the macromolecule under a particular controlledenvironmental condition, but not when the environmental conditions arechanged. Such conditions can be, for example, but not limited to, changein [H⁺], change in environmental temperature, change in stringency,change in hydrophobicity, change in hydrophilicity, or the like.

In another embodiment, the invention provides a compound, wherein thecompound further comprises a linker molecule, the linker moleculeselected from the group consisting of a thiol group, a sulfide group, aphosphate group, a sulfate group, a cyano group, a piperidine group, anFmoc group, and a Boc group. In another embodiment the compound isselected from the group consisting of a bifunctional alkyl sulfide andgold.

BRIEF DESCRIPTION OF THE DRAWINGS

Provided as embodiments of this disclosure are drawings which illustrateby exemplification only, and not limitation, wherein:

FIG. 1 shows schematic of a two-chamber dual-pore device along with adual-amplifier electronics layout for independent voltage control (V₁,V₂) and current measurement (I₁,I₂) of each pore. Chambers A and B arevolumetrically separated except through the dual-pore channel, comprisedof a ˜30 nm diameter channel drilled through SiN-metal-SiN layers, and ametallic membrane with a 2-10 nm nanopore;

FIG. 2 shows that, electrically, V₁ drops across resistance R₁associated with resistance of the −30 nm channel path through top SiNmembrane, and V₂ drops across R₂ which is dominated by the resistance ofthe smaller nanopore in the metallic membrane. Shrinking the top porecan increase R₁ until R₁≈R₂. The metal layer (a continuous sheet ofchrome or silver for example) will serve as a grounded electrode surfacein the channel; and

FIG. 3 illustrates that a DNA molecule captured in the dual-pore channelcan be electrophoretically controlled by competing voltages, and ionic(KCl) current through the metallic membrane pore can be examined forsingle-nucleotide sensitivity. This figure also shows closure of the topnanopore (2-10 nm diameter) to provide a two-pore configuration that mayimprove control. Target chip parameter ranges include: 10≤h≤50 nm,50≤h_(m)≤100 nm, t=10 nm, dual-pore channel diameter: 20-100 nm. Arrowsshow direction of voltage forces during electrophoretic control, with|V₁|=|V₂|+δV, and small δV>0 (<0) for tunable, net motion in thedirection of chamber A (B).

Some or all of the figures are schematic representations forexemplification; hence, they do not necessarily depict the actualrelative sizes or locations of the elements shown. The figures arepresented for the purpose of illustrating one or more embodiments withthe explicit understanding that they will not be used to limit the scopeor the meaning of the claims that follow below.

DETAILED DESCRIPTION

Throughout this application, the text refers to various embodiments ofthe present nutrients, compositions, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present invention.

Also throughout this disclosure, various publications, patents andpublished patent specifications are referenced by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications are hereby incorporated by reference into thepresent disclosure to more fully describe the state of the art to whichthis invention pertains.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an electrode” includes a plurality ofelectrodes, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thedevices and methods include the recited components or steps, but notexcluding others. “Consisting essentially of” when used to definedevices and methods, shall mean excluding other components or steps ofany essential significance to the combination. “Consisting of” shallmean excluding other components or steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

All numerical designations, for example, distance, size, temperature,time, voltage and concentration, including ranges, are approximationswhich are varied (+) or (−) by increments of 0.1. It is to beunderstood, although not always explicitly stated that all numericaldesignations are preceded by the term “about”. It also is to beunderstood, although not always explicitly stated, that the componentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

Device for Detecting Polymers

One embodiment of the present disclosure provides a device that includesa channel through and defined by a plurality of layers surrounding thechannel, the channel connecting a first and a second chambers separatedby the plurality of layers. Accordingly, the channel enables fluidcommunication between the chambers. Further, the channel is comprised ofregions that are defined by the layers surrounding these regions.

In one aspect, the plurality of layers include at least a first layer, asecond layer, and a conductive middle layer disposed between the firstand second layers. The region defined by the first layer is denoted asan inlet (or alternatively a “first pore”) and the region defined by thesecond layer is denoted as an outlet (or alternatively as a “secondpore”). It is noted that the terms inlet and outlet are relative termsand thus do not require that, for instance, fluid must enter the inletand come out of the outlet. Collectively, such a device is also termed a“two-chamber dual-pore device.”

In one aspect, the first and second chambers and the middle layer areconnected to a power supply. As such, voltages can be establishedbetween the first chamber and the middle layer and between the middlelayer and the second chamber. In some aspects, these two voltages areindependently adjustable.

FIG. 1 illustrates such a device, which includes a channel 120surrounded by a number of layers including a first layer (104), a secondlayer (101) and a middle layer (102) between the first and second layer.In this example, an additional layer (103) is provided between the firstlayer and the middle layer, which is made of the same material as thesecond layer 101. Here, the second layer 101 and layer 103 are both madeof silicon whereas the middle layer 102 is metal. The first layer 104 isa metallic membrane. As the layer 103 is not conductive, the first layer104 and the middle layer 102 insulated from each other.

The channel 120 can be considered to be comprised of multiple regions,each region defined by the surrounding layer. In this context, theportion of the channel defined by the first layer 104 can be denoted asan inlet (105), and the portion of the channel defined by the secondlayer 101 can be denoted as an outlet (106).

The device in FIG. 1 further includes two chambers, 107 (“Chamber A”)and 108 (“Chamber B”), which have fluid communication through thechannel 120 only. The chambers can be formed with walls (for example,112 and 113) that are comprised of materials such as silicon.

Diameters, depths and distance of the inlet and outlet of the device canbe adjusted for particular use of the device. For instance, in oneaspect, the inlet has a diameter that is about 0.5 nm to about 100 nm.In another aspect, the diameter is at least about 0.5, or 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40 or 50 nm. In another aspect, the diameter is notgreater than about 100, or 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15,or 10 nm. In one aspect, the inlet is about 1 nm to about 20 nm indiameter.

In one aspect, the outlet has a diameter that is about 0.5 nm to about100 nm. In another aspect, the diameter of the outlet is at least about0.5, or 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 nm. In anotheraspect, the diameter is not greater than about 100, or 90, 80, 70, 60,50, 40, 35, 30, 25, 20, 15, or 10 nm. In one aspect, the outlet is about1 nm to about 20 nm in diameter. In some aspects, the outlet's diameteris greater than that of the inlet.

In one aspect, the inlet has a depth that is from about 0.1 nm to about100 nm. In one aspect, the depth of the inlet is at least about 0.1 or0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 10, 15 or 20nm. In another aspect, the depth is not greater than about 100, or 90,80, 70, 60, 50, 40, 35, 30, 25, 20, 15, or 10 nm. In one aspect, theinlet is about 0.2 nm to about 10 nm in depth.

In one aspect, the outlet has a depth that is from about 0.1 nm to about100 nm. In one aspect, the depth of the outlet is at least about 0.1 or0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 10, 15 or 20nm. In another aspect, the depth is not greater than about 100, or 90,80, 70, 60, 50, 40, 35, 30, 25, 20, 15, or 10 nm. In one aspect, theoutlet is about 0.2 nm to about 10 nm in depth.

In some aspects, the inlet and the outlet are about 10 nm to about 1000nm apart from each other. Such a distance, in one aspect, is at leastabout 10, or 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm and, in anotheraspect, is not greater than about 1000, or 900, 800, 700, 600, 500, 400,300, 200 or 100 nm.

In some aspects, the inlet has a substantially round shape.“Substantially round”, as used here, refers to a shape that is at leastabout 80 or 90% in the form of a cylinder. In some embodiments, the poreis square, rectangular, triangular, oval, or hexangular in shape.

It is shown that each of the two chambers and the middle layer of thedevice is connected to a power supply. The power supply 114, as shown,is connected to the middle layer 102 at electrode 111, to Chamber A atelectrode 109 and to Chamber B at electrode 110. In one aspect, themiddle layer is grounded, and the voltages rendered between the middlelayer and each of the two chambers is independently adjustable.

An exemplary setup for generation and adjustment of the voltages isprovided in FIG. 2. Electrically, V₁ drops across resistance R₁ (betweenChamber A and the middle layer) associated with resistance of the −30 nmchannel path through top SiN membrane, and V₂ drops across R₂ (betweenChamber B and the middle layer) which is dominated by the resistance ofthe inlet, which in this case has a smaller diameter than the outlet.

In some aspects, the power supply is comprised of a voltage-clamp or apatch-clamp for supplying the voltage across each pore, which can alsomeasure the current through each pore independently. In this respect,the power supply can set the middle layer to a common ground for bothvoltage sources. In one aspect, the power supply is configured toprovide a first voltage between one chamber (for example, Chamber A inFIG. 1) and the middle layer, and a second voltage between the secondchamber (for example, Chamber B in FIG. 1) and the middle layer.

Adjustment of the voltages can be used to control the movement ofcharged particles in the channel. For instance, when both voltages areset in the same direction, a properly charged particle can be moved fromthe one chamber to the middle of the channel and to the other chamber,or the other way around, sequentially. Otherwise, a charged particle canbe moved from either of the chambers to the middle of the channel andkept there.

The adjustment of the voltages in the device can be particularly usefulfor controlling the movement of a large molecule, such as a chargedpolymer, that is long enough to cross the entire channel at the sametime. In such an aspect, the movement and the rate of movement of themolecule can be controlled by the relative magnitude and direction ofthe voltages, which will be further described below.

The device can contain materials suitable for holding liquid samples, inparticular, biological samples, and/or materials suitable fornanofabrication. In one aspect, such materials include dielectricmaterials such as, but not limited to, silicon, silicon nitride, silicondioxide, graphene, carbon nanotubes, TiO₂, HfO₂, Al₂O₃, or othermetallic layers, or any combination of these materials. A single sheetof graphene forms a membrane ˜0.3 nm thick, and can be used as thepore-bearing membrane, for example.

Fabrication of the Device

The present disclosure, in one embodiment, provides an integratedtwo-chamber dual-pore device, which can be smaller than the size of adesktop PC, and will enable long-read (>10 kbp) nanopore sequencing. Inone aspect, the device is based on the dual-pore configuration thatincorporates a pair of vertically-aligned pores drilled in parallelsubstrates, with each pore independently controlled using a dualamplifier architecture (FIG. 1). Such a device leverages an integratedamplifier and microfluidic devices incorporating solid-state nanopores.

It is contemplated that such a design can reduce and control the rate ofDNA passage through the nanopores, while still generating asequence-sensitive current.

Chip fabrication and nanopore fabrication can be done using thin filmdeposition and etching along with focused ion beam (FIB) milling.Electron beams can also be used, in addition to ion beams, to drillnanopores and channels. A silicon substrate can serve as a template andthin films of metal and dielectric material (SiO₂ and silicon nitride)can be deposited using evaporation and chemical vapor deposition.Metallic materials include chrome (Cr), aluminum (Al), platinum (Pt),gold (Au), or any other metal that can be deposited with contemporarythin film deposition methods such as e-beam evaporation, thermalevaporation, MBE (molecular beam epitaxy), or ALD (atomic layerdeposition).

Photolithography and chemical etching can be used to form hollow spacesfor fluid entry and electrical access to metallic layers. A micropore (4μm diameter) can first be opened through reactive etching and then aninitial nanopore (˜30 nm diameter) can be drilled using a FIB. Thisprocess can be successfully been used to define nanopores in ˜100 nmthin dielectric membranes.

Active feedback can allow the drilling of a second, smaller nanopore(2-10 nm) in a thin metallic layer, forming the most significant sensingaperture (see FIG. 1). Both nanopores can be sculpted (shrunk) to thedesired size individually by applying the correct beam focus to eachlayer (FIG. 3), and this method can be used for improved sensing andcontrol. Having both pores 2-10 nm can facilitate detangling of longssDNA, for example, or enable cross-correlation of the recorded ioniccurrents (pores of common diameters have comparable signal-to-noiseratios).

The 10 nm metallic membrane can be refined for single-nucleotidesensitivity by thinning the membrane near the site of nanopore drilling.FIB-sculpting the membrane near the pore can achieve 1-2 nm thickness,followed by FIB-drilling the pore to 2-5 nm diameter. Direct sequencingof long (>10 kbp) single-stranded DNA can be attempted. The ssDNA willform a large amount of secondary structure, which must be unravelled fornanopore sensing and control. It has been shown that this is achievable,using a single 8 nm nanopore in a Si-membrane.

As an alternative to the metallic membrane, one can incorporate agraphene layer into the chip. Incorporating graphene will be morecomplex process, but the membrane is durable and likely to providesingle-nucleotide sensitivity. In particular, graphene has been shown tofunction as an insulating membrane, and with a drilled nanopore, capableof detecting capture and passage of DNA. Researchers have shown thatdrilling nanopores in multi-layered graphene membranes results in aterrace effect, enabling the use of thicker and thus sturdier membranesthat are thin near the pore. Numerical studies also support that ioniccurrent through a 2.5 nm graphene pore would be single-nucleotidesensitive (Venkatesan and Bashir, Nature Nanotechnology, 6:615-624,2011), but no experiment has shown this due (in part) to the lack of aDNA rate control method that is graphene compatible.

Controlling Movement of Molecules with the Device

By virtue of the voltages present at the inlet and outlet of the device,charged molecules can be moved through the pores between chambers. Speedand direction of the movement can be controlled by the magnitude anddirection of the voltages. Further, because each of the two voltages canbe independently adjusted, the movement and speed of a charged moleculecan be finely controlled in each chamber and through the channel.

One example concerns a charged polymer, such as a polynucleotide, havinga length that is longer than the distance between the two pores. Forexample, a 1000 bp dsDNA is ˜340 nm in length, and would besubstantially longer than a channel that include an inlet and an outlet,each being 10 nm in depth and 100 nm apart. In a first step, thepolynucleotide is loaded into either of the two chambers. By virtue ofits negative charge under a physiological condition (˜pH 7.4), thepolynucleotide can be moved across the channel. Therefore, in a secondstep, two voltages, in the same direction and at the same or similarmagnitudes, are applied to the channel to induce movement of thepolynucleotide across both the inlet and the outlet sequentially.

At about time when the polynucleotide reaches the end of the channel,one or both of the voltages can be changed. Since the distance betweenthe inlet and the outlet is selected to be shorter than the length ofthe polynucleotide, when the polynucleotide reaches the end of thechannel, it spans the entire channel. A prompt change of direction ofthe voltage at the first pore, therefore, will generate a force thatpulls the polynucleotide straight, across the channel (illustration inFIG. 3).

If, at this point, the magnitude of the voltage-induced force at oneside is less than that of the voltage-induced force at the other side ofthe channel, then the polynucleotide will continue crossing the channel,but at a lower speed. In this respect, it is readily appreciated thatthe speed and direction of the movement of the polynucleotide can becontrolled by the directions and magnitudes of both voltages. As will befurther described below, such a fine control of movement has broadapplications.

Accordingly, in one aspect, provided is a method for controlling themovement of a charged polymer through a channel. The method entails (a)loading a sample comprising a charged polymer in one of the first orsecond chamber of the device of any of the above embodiments, whereinthe device is connected to a voltage-clamp or patch-clamp system forproviding a first voltage between the first chamber and the middlelayer, and a second voltage between the middle layer and the secondchamber; (b) setting an initial first voltage and an initial secondvoltage so that the polymer moves through the chambers, thereby locatingthe polymer across both the inlet and outlet; and (c) adjusting thefirst voltage and the second voltage so that both voltages generateforce to pull the charged polymer away from the middle layer(voltage-competition mode), wherein the two voltages are different inmagnitude, under controlled conditions, so that the charged polymermoves through the channel and both the inlet and the outlet in onedirection and in a controlled manner.

For the purpose of establishing the voltage-competition mode in step(c), the relative force exerted by each voltage at each pore is to bedetermined for each two-pore device used, and this can be done withcalibration experiments by observing the influence of different voltagevalues on the motion of the polynucleotide (motion can be measured bysensing location-known and detectable features in the polynucleotide,with examples of such features detailed later in this document). If theforces are equivalent at each common voltage, for example, then usingthe same voltage value at each of the inlet and outlet (with commonpolarity in both chambers relative to grounded middle layer) creates azero net motion in the absence of thermal agitation (the presence andinfluence of Brownian motion is discussed below). If the forces are notequivalent at each common voltage, then achieving equal forcing requiresidentification and use of a larger voltage at the inlet or outlet thatexperiences a weaker force at the common voltage. Calibration forvoltage-competition mode is required for each two-pore device, and wouldbe required for specific charged polymers or molecules for whichfeatures that pass through each pore influence the force.

In one aspect, the sample containing the charged polymer is loaded intothe first chamber and the initial first voltage is set to pull thecharged polymer from the first chamber to the middle of the channel andthe initial second voltage is set to pull the polymer from the middle ofthe channel to the second chamber. Likewise, the sample can be initiallyloaded into the lower chamber.

In another aspect, the sample containing the charged polymer is loadedinto the channel and the initial first voltage is set to pull thecharged polymer from the channel to the first chamber and the initialsecond voltage is set to pull the charged polymer from the channel tothe second chamber.

The term “charged polymer” or “polymer” refers to a polymer thatcontains sufficient charged units at the pH of the solution that it canbe pulled through a pore by electrostatic forces. In one embodiment,each unit of the charged polymer is charged at the pH selected. Inanother embodiment, the charged polymer is comprised of sufficientcharged units to be pulled into and through the pores by electrostaticforces. For example, a peptide containing sufficient entities which canbe charged at a selected pH (lysine, aspartic acid, glutamic acid, etc.)so as to be used in the devices and methods described herein is acharged polymer for purposes of this invention. Likewise, a copolymercomprising methacrylic acid and ethylene is a charged polymer for thepurposes of this invention if there is sufficient charged carboxylategroups of the methacrylic acid residue to be used in the devices andmethods described herein is a charged polymer for purposes of thisinvention. In one embodiment, the charged polymer is comprised one ormore charged units at or close to one terminus of the polymer. Inanother embodiment, the charged polymer is comprised of one or morecharged units at or close to both termini of the polymer.

In some aspects, the charged polymer is a polynucleotide or apolypeptide. In a particular aspect, the charged polymer is apolynucleotide. Non-limiting examples of polynucleotides includedouble-stranded DNA, single-stranded DNA, double-stranded RNA,single-stranded RNA, and DNA-RNA hybrids.

In one aspect, the adjusted first voltage and second voltage at step (c)are about 10 times to about 10,000 times as high, in magnitude, as thedifference between the two voltages. For instance, the two voltages are90 mV and 100 mV, respectively. The magnitude of the voltages (˜100 mV)is about 10 times of the difference between them, 10 mV. In someaspects, the magnitude of the voltages is at least about 15 times, 20times, 25 times, 30 times, 35 times, 40 times, 50 times, 100 times, 150times, 200 times, 250 times, 300 times, 400 times, 500 times, 1000times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000times, 8000 times or 9000 times as high as the difference between them.In some aspects, the magnitude of the voltages is no more than about10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times,4000 times, 3000 times, 2000 times, 1000 times, 500 times, 400 times,300 times, 200 times, or 100 times as high as the difference betweenthem.

In one aspect, real-time or on-line adjustments to first voltage andsecond voltage at step (c) are performed by active control or feedbackcontrol using dedicated hardware and software, at clock rates up tohundreds of megahertz. Automated control of the first or second or bothvoltages is based on feedback of the first or second or both ioniccurrent measurements.

Sensor in the Device

The device of the present disclosure can be used to carry out analysisof molecules or particles that move or are kept within the device. Inone aspect, the device further includes one or more sensors to carry outthe analysis. In one aspect, the sensor includes a pair of electrodesplaced at two sides of the inlet or the outlet to measure an ioniccurrent across the inlet or the outlet when a molecule or particle, inparticular a polymer, moves through.

In an alternative aspect, when a single sensor in used, it iscontemplated that the single sensor preferably does not include such apair of ionic current sensors. In one aspect, the device includes asensor suitable for identifying a polymer, or individual components of apolymer. Non-limiting examples of individual components include monomerunits and monomer units with modifications approximate to or on themonomer unit. When the polymer is a polynucleotide, an individualcomponent can be one or more nucleotide units or the nucleotide unitsbound by a protein factor, without limitation.

The sensors used in the device can be any sensor suitable foridentifying a molecule or particle, such as a polymer. For instance, asensor can be configured to identify the polymer by measuring a current,a voltage, pH, an optical feature or residence time associated with thepolymer or one or more individual components of the polymer.

In one embodiment, the sensor measures an optical feature of the polymeror a component (or unit) of the polymer. One example of such measurementincludes identification by infrared (or ultraviolet) spectroscopy of anabsorption band unique to a particular unit.

When residence time measurements are used, they will correlate the sizeof the unit to the specific unit based on the length of time it takes topass through the sensing device.

Still further, the sensor can include an enzyme distal to that sensorwhich enzyme is capable of separating the terminal unit of the polymerfrom the penultimate unit thereby providing for a single molecular unitof the polymer. The single molecule, such as a single nucleotide or anamino acid, can then be detected with methods such as mass spectrometry.Methods for measuring such a single unit are known in the art andinclude those developed by Caltech (see, for example,http://spectrum.ieee.org/tech-talk/at-work/test-and-measurement/a-scale-for-weighing-single-molecules).The results of that analysis can be compared to those of the sensingdevice to provide confirmation of the correct analysis.

Accordingly, the sensor can be placed at a location within the devicethat enables such measurement. In one aspect, the sensor can include twoelectrodes and be placed in either or both the inlet and outlet. Whenthe polymer moves across the channel, the two electrodes are then onboth sides of the polymer and thus can identify an individual componentby measuring a current or a voltage, or the change thereof, across theindividual component.

In yet another aspect, the sensor does not include electrodes but takesa form of a membrane or scaffold having an opening (for example, a hole)that allows passing of a polymer. For instance, one or more layers ofgraphene membrane with an opening, which can be just like a nanopore asthe other nanopores, can act as the auxiliary sensor, which itself ispositioned in the chamber between the inlet and outlet. In one aspect,the graphene member comprises a single sheet, double sheet, or more thantwo sheets.

In some embodiments, the sensor is configured to proximate a polymerwhen the polymer is loaded in both the first and the second pores.Therefore, when the polymer moves through the channel, the sensor isclose enough to the polymer to measure the polymer.

In some embodiments, the sensor is configured to form a tunnel gapallowing a polymer to pass through the tunnel gap. When a polymer movesthrough the tunnel gap, the sensor is then able to identify theindividual components of the polymer. It has been shown that individualnucleotides can be discriminated in a precision 0.8 nm tunneling gap.

In some embodiments, the sensor is functionalized with reagents thatform distinct non-covalent bonds with each DNA base. In this respect,the gap can be larger and still allow effective measuring. For instance,a 2.5 nm gap can be as effective, when used with a functionalized, as a0.8 nm gap. Tunnel sensing with a functionalized sensor is termed“recognition tunneling.” Using a Scanning Tunneling Microscope (STM)with recognition tunneling, a DNA base flanked by other bases in a shortDNA oligomer can be identified.

Recognition tunneling can also provide a “universal reader” designed tohydrogen-bond in a unique orientation to each of the four DNA bases (A,C, G, T) and also to the base 5-methyl-cytosine (mC) which is naturallyoccurring due to epigenetic modifications.

A limitation with the conventional recognition tunneling is that it candetect only freely diffusing DNA that randomly binds in the gap, or thathappens to be in the gap during microscope motion, with no method ofexplicit capture to the gap. Further, the collective drawbacks of theSTM setup will go away when the recognition reagent, once optimized forsensitivity, is incorporated within an electrode tunneling gap in ananopore channel.

Accordingly, in one embodiment, the sensor comprises surfacemodification by a reagent. In one aspect, the reagent is capable offorming a non-covalent bond with a nucleotide. In a particular aspect,the bond is a hydrogen bond. Non-limiting examples of the reagentinclude 4-mercaptobenzamide and 1-H-Imidazole-2-carboxamide.

A significant advantage of the methods in the present technology is thatis that they can be engineered, in principle, to provide direct trackingof progress through homopolymeric regions (base repeats). Direct baserepeat tracking is not possible with ionic current sensing. Trackingrepeats is essential, for example, since deletions and insertions ofspecific mononucleotide repeats (7, 9 nt) within human mitochondrial DNAhave been implicated in several types of cancer.

In ionic current sensing, there is no distinct signal-per-nucleotide ofmotion of homopolymeric ssDNA through the pore. It is contemplated thatan ideal nanopore sequencing platform should utilize an auxiliarysensing method that can track per-nucleotide motion progress while alsoachieving single-nucleotide sensitivity. Transitions between neighboringnucleotides in oligomers can be observable with recognition tunneling,making it a candidate for sequencing that permits direct base-repeattracking.

Therefore, the methods of the present technology can provide DNAdelivery rate control for one or more recognition tunneling sites, eachpositioned in one or both of the nanopore channels, and voltage controlcan ensure that each nucleotide resides in each site for a sufficientduration for robust identification.

Sensors in the devices and methods of the present disclosure cancomprise gold, platinum, graphene, or carbon, or other suitablematerials. In a particular aspect, the sensor includes parts made ofgraphene. Graphene can act as a conductor and an insulator, thustunneling currents through the graphene and across the nanopore cansequence the translocating DNA.

In some embodiments, the tunnel gap has a width that is from about 1 nmto about 20 nm. In one aspect, the width of the gap is at least about 1nm, or alternatively at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6,7, 8, 9, 10, 12 or 15 nm. In another aspect, the width of the gap is notgreater than about 20 nm, or alternatively not greater than about 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nm. Insome aspects, the width is between about 1 nm and about 15 nm, betweenabout 1 nm and about 10 nm, between about 2 nm and about 10 nm, betweenabout 2.5 nm and about 10 nm, or between about 2.5 nm and about 5 nm.

Characterization of a Polymer in the Device

A polymer, such as a DNA molecule, can be analyzed in the device of thepresent disclosure. In one aspect, the polymer is loaded into thechannel in the device, as described above. Once the polymer is loaded,it is at a position suitable for detection by the sensor, by means ofmeasuring a current, a voltage, pH, an optical feature or residence timeassociated with the polymer or components of the polymer.

For instance, a polynucleotide can be loaded into the channel by twovoltages having the same direction. In this example, once the directionof the voltage applied at the first pore (either of the inlet or outlet)is inversed and the new voltage-induced force is slightly less, inmagnitude, than the voltage-induced force applied at the second pore(the other of the inlet or outlet), the polynucleotide will continuemoving in the same direction, but at a markedly lower speed. In thisrespect, the amplifier supplying voltage across the second pore alsomeasures current passing through the second pore, and the sensor thendetermines the identification of a nucleotide that is passing throughthe pore, as the passing of each different nucleotide would give rise toa different current signature (for example, based on shifts in the ioniccurrent amplitude). Identification of each nucleotide in thepolynucleotide, accordingly, reveals the sequence of the polynucleotide.

In some aspects, repeated controlled delivery for re-sequencing apolynucleotide further improves the quality of sequencing. Each voltageis alternated as being larger, for controlled delivery in eachdirection. Also contemplated is that the two currents through the twopores can be correlated to improved accuracy. It is contemplated thatBrownian motion may cause fluctuations in the motion of a molecule,affecting controlled delivery of the molecule. Such an effect, however,can be minimized or avoided by, for example, during DNA sequencing,repeated controlled delivery of the DNA and averaging the sequencingmeasurements. Still further, it is contemplated that the impact ofBrownian motion on the controlled motion of large molecules, such aspolynucleotides and polypeptides, would be insignificant in particularwhen competing forces are pulling the largest molecules apart,generating tension within the molecule. It is contemplated that adhesionof the DNA to the pore walls, by surface charge modifications orchemistry to the pore surface, to create friction can also mitigate theinfluence of Brownian motion on the control performance of the two poremethod.

Such a method provides a ready solution to problems that have not beensolved in the prior art.

For instance, it is known that there are two competing obstacles toachieve the controlled delivery and accurate sensing required fornanopore sequencing. One is that a relatively high voltage is required,at the pore, to provide enough sequencing sensitivity. On the otherhand, high voltages lead to fast passing of a polynucleotide through thepore, not allowing sufficient time for identification of eachnucleotide.

More specifically, the nanopore sequencing platform requires that therate of polynucleotide passage through the pore be controlled to 1ms/nucleotide (nt) or slower, while still generating asequence-sensitive current. This requires sufficiently highsignal-to-noise for detecting current signatures (high voltage isbetter), but sufficiently slow motion of the molecule through the poreto ensure measurements are within the recording bandwidth (low voltageis better). In single pore implementations, polynucleotide speed isproportional to voltage, so higher voltage is better for sensing butworse for reducing polynucleotide speed: rates are 1 μs/nt and faster(>1000 times too fast) at voltages that promote polynucleotide capture.On the other hand, lower voltages reduce sensing performance, and alsoincrease the relative contribution of rate fluctuations caused byBrownian motion that will undermine read accuracy.

Other than what is described herein, there are currently no methods foraddressing these obstacles that do not involve the use of enzymes oroptics, both of which work only in specialized nanopore techniques.

Several approaches have been proposed to address the problem associatedwith the lack of sensing capability, and under low voltages. One is toengineer biological nanopores to improve their sensitivity. Another isto use graphene membranes, which as a single sheet are thinner than thedistance between nucleotides in ssDNA. Still another is the use of anauxiliary current measured in close proximity to the nanopore (forexample, tunneling currents).

Biological nanopores have been tested in the first approach. Theα-hemolysin nanopore is the most commonly used biological pore inresearch. Studies have shown that α-hemolysin can resolve singlenucleotide substitutions in homopolymers and abasic (1′,2′-dideoxy)residues within otherwise all-nucleobase DNA. However, single nucleotidesensitivity is not possible in heteromeric DNA with wild-type (WT)α-hemolysin, for which the ionic current is influenced by ˜10nucleotides in the channel. Protein engineering of α-hemolysin has beenused to improve its sensitivity for DNA analysis and sequencing. Onesuch mutant pore uses α-hemolysin with a covalently attached molecularadapter (Clarke et al., Nat. Nanotech, 4(4):265-70, 2009) that iscapable of discriminating the four nucleoside 5′-monophosphate moleculeswith high accuracy. However, this mutant pore does not appear to havesensitivity for sequencing intact heteromeric ssDNA that passes throughthe pore.

Another exemplary biological pore is MspA, which has a funnel-like shapethat focuses the sensitivity of the ionic current to the bottom of thechannel. Moreover, achieving rate reduction of DNA through MspA andα-hemolysin can be achieved by using enzymes. As shown in FIG. 1 of(Manrao et al., Nature Biotechnology, 30:349-53, 2012), rate reductionof DNA through is achieved with the enzyme perched on the MspA nanopore.However, this results in non-deterministic sensing durations, repeatedreads induced by backtracking, and an inability to sense homopolymericregions. The mechanism of phi29 polymerase mediated DNA translocationwas developed in (Cherf et al., Nat. Biotech., 30(4):344-8, 2012) onα-hemolysin and implemented on the more sensitive MspA nanopore (Manraoet al., Nature Biotechnology, 30:349-53, 2012). Step-wise rates ofpolymerization-catalyzed translocation are 2.5-40 nt/s, meeting therequirements for DNA rate reduction. However, while enzymes on bioporescan reduce the rate of translocation, there is lack of control over thedwell time of each nucleotide, which will make blind tracking of repeatsvery difficult, and challenging to differentiate from long pauses on asingle nucleotide read. In terms of sensitivity, as shown in FIG. 3 of(Manrao et al., Nature Biotechnology, 30:349-53, 2012), reading arepetitive DNA template can be achieved with phi29 on MspA. The FIG. 3a) shows an example trace for a DNA template composed of repeated CATtrinucleotides, with the exception of one CAG triplet in the middle ofthe sequence. The * represent “toggles” detected (by human analysis) asrepeated transitions between two levels, which are intrinsic to theenzyme-based control method and incur read errors. The FIG. 3b ) alsoshows Idealization of (3 a), with mean currents of levels aligned withthe known DNA sequence, and removing disparity of measured durationsshown in (3 a). Idealization shows a repeating pattern of three levelsinterrupted by the single dG substitution. Four levels are affected bythe single dG with the largest deviations closest to the substitution,suggesting residual current is principally influenced by ˜4 nucleotides.That the ionic current through MspA is influenced by ˜4 nucleotides mostproximal to the limiting aperture adds considerable complexity toidentifying the sequence. While one would ideally build a library ofdistinct current amplitudes that map to each of the 4⁴=256 combinations,as suggested in the art, such a library will be difficult to achieve.The reason is that identifying step transitions in channel currentrecordings requires a signal-to-noise ratio (SNR) of at least 2 withhalf-amplitude methods (SNR 1.5 for Markov-based methods). With RMSnoise of 0.5 pA at recording bandwidths, amplitudes shifts must be atleast 1 pA to have the required SNR, resulting in only ˜40 detectablelevels within the amplitude range of 40 pA with MspA (or, at most 53levels at SNR 1.5). Moreover, fewer than 40 levels will be observedsince the range will not be uniformly utilized, and while furtherfiltering could reduce noise to add amplitude discrimination it alsoresults in missing more of the faster ssDNA motion transitions that arealready present.

Presently, there is no nanopore for which ionic current sensing canprovide single-nucleotide sensitivity for nucleic acid sequencing.Still, improvements to the sensitivity of biological pores andsolid-state pores (graphene) are active and ongoing research fields. Oneissue is that ionic current sensing does not permit direct tracking ofprogress through homolymeric regions (base repeats), since there is nodistinct signal-per-nucleotide of motion of homopolymeric ssDNA throughthe pore. Tracking repeats is essential, for example, since deletionsand insertions of specific mononucleotide repeats (7, 9 nt) within humanmitochondrial DNA have been implicated in several types of cancer(Sanchez-Cespedes, et al., Cancer Research, 61(19):7015-7019, 2001).While enzymes on biopores can reduce the rate of translocation, there islack of control over the dwell time of each nucleotide. On the otherhand, using a constant delivery rate with two-pore control,non-deterministic pauses are eliminated, and accurate estimation ofrepeat lengths can be made. Re-reading the repeat section many times canalso improve the estimation errors and identify error bounds, and thiscan be done without having to reverse the polymerization chemistrycaused by enzymes.

A recent study showed that, with a single nanopore, reduced rates cannotbe achieved by merely reducing the voltage (Lu et al., BiophysicalJournal, 101(1):70-9, 2011). Instead, as voltage is reduced, the ratesof a single-stranded DNA (ssDNA) become more random (includingbacktracking), since Brownian motion becomes an increasing contributorto velocity fluctuations. The study also shows that high voltage forceis required to suppress Brownian-motion induced velocity fluctuationsthat will otherwise confound sequencing measurements, even when using anidealized single-nucleotide-sensitive nanopore sensor.

The sequencing method provided in the present disclosure, based on atwo-pore device, provides a ready solution to these problems andadditional advantages over the existing methods. In concert with one ortwo pores that have sufficient sensitivity for sequencing, at high orlow voltage, the two pore control solves the sequencing rate controlproblem of single nanopore implementations. Such pores can includebiological pores housed in solid-state substrates, biological pores inmembranes formed across solid-state substrates, or solid-state pores(for example, in graphene, silicon, or other substrates). In one aspect,an enzyme such as phi29 on a biological pore such as MspA can be used atone or both pores, with high voltages used to generate large signals forsequencing and a low differential voltage that generates a force on eachenzyme that is sufficient to hold the enzymes in position atop each poreand permit polymerization-catalyzed DNA motion, but not large enough tostall or dissociate the enzymes. Such a configuration can improve themethods in Cherf et al., Nat. Biotech., 30(4):344-8, 2012 and Manrao etal., Nature Biotechnology, 30:349-53, 2012, by significantly boostingthe measurement signal, and permitting two pores to read one stand ofDNA at the same time.

In addition, the method of the present disclosure can generatesufficiently high voltage at the pore to ensure detection sensitivity atthe pore using ionic current sensing. It is plausible that high voltagewould suppress Brownian motion enough to ensure constant rates througheach pore, and configuration of the DNA outside each pore will affectthe energetics of motion of DNA in either direction. Additionally, thevoltage competition used in the method (FIG. 3) can be tuned so that themolecule spends sufficient time in the pore to allow analysis of themolecule. Still further, the present method is free of the need forenzymes, optics, or attachments to the DNA. Therefore, the methodprovides high signal-to-noise detection currents through the nanoporewhile regulating the molecule delivery rate, a capability that is notpossible with single nanopore implementations.

The method can be used to identify the composition of monomers in acharged polymer. In one aspect, the monomer unit is a nucleotide whenthe polymer is a single stranded DNA or RNA. In another aspect, themonomer unit can be a nucleotide pair, when the polymer is doublestranded.

In one aspect, the method can be used to identify a modification to thepolymer, such as a molecule bound to a monomer, in particular when thebound molecule is charged. The bound molecule does not have to becharged, however, as even a neutral molecule can change the ioniccurrent by virtue of its size.

In another aspect, the modification comprises the binding of a moleculeto the polymer. For instance, for a DNA molecule, the bound molecule canbe a DNA-binding protein, such as RecA, NF-κB and p53. In yet anotheraspect, the modification is a particle that binds to a particularmonomer or fragment. For instance, quantum dots or fluorescent labelsbound to a particular DNA site for the purpose of genotyping or DNAmapping can be detected by the device. Accordingly, the device of thepresent disclosure provides an inexpensive way for genotyping and DNAmapping as well, without limitation.

In one aspect, the polymer is attached to a solid support, such as abead, at one end of the polymer.

Also provided, in one embodiment, is a method for determining thesequence of a polynucleotide, comprising: loading a sample comprising apolynucleotide in one of the first or second chamber of the device ofany of the above embodiments, wherein the device is connected to avoltage-clamp or patch-clamp system for providing a first voltagebetween the first chamber and the middle layer, and a second voltagebetween the middle layer and the second chamber; (b) setting an initialfirst voltage and an initial second voltage so that the polynucleotidemoves through the chambers, thereby locating the polymer across both theinlet and outlet; (c) adjusting the first voltage and the second voltageso that both voltages generate force to pull the polynucleotide awayfrom the middle layer, wherein the two voltages are different inmagnitude, under controlled conditions, so that the polynucleotide movesthrough the channel and both the inlet and the outlet in one directionand in a controlled manner; and (d) identifying each nucleotide of thepolynucleotide that passes through the inlet or the outlet, when thenucleotide passes that inlet or outlet.

EXAMPLES

The present technology is further defined by reference to the followingexamples. It will be apparent to those skilled in the art that manymodifications, both to devices and methods, may be practiced withoutdeparting from the scope of the current invention.

Example 1. Capture and Control of Individual dsDNA Molecules in Pores

This example shows that capture of DNA into each pore in a two-chamberdual-pore device is readily detected as shift in each independent ionicpore current measured.

This example demonstrates dual-pore capture using dsDNA with and withouta bead attached to one end. Experiments with bead-tethered ssDNA canalso be explored.

Upon capture and stalling of the DNA, the pore voltage nearest the beadcan be reversed and increased until the competing force on the DNA drawsit back toward the other chamber. The ionic current in either pore(inlet or outlet) can readily detect capture and exit of the DNA duringthe experiment.

When a bead is used, the bead has a proper size that prevents the beadfrom passing the channel. Methods that ensure a 1 to 1 bead-DNA ratiohave been developed in the art. For example, monovalentstreptavidin-coated Quantum dots (QDs; QD655, Invitrogen) conjugated tobiotinylated DNA duplexes (or ssDNA) can provide beads in the 20-30 nmdiameter range, with larger beads (30-100 nm) possible by using goldparticles or latex. The influence of bead on hydrodynamics and charge,as it relates to capture rate, can be considered in designing theexperiments.

Without beads, dsDNA passes through a pore at ˜0.1 ms/kbp. DNA oflengths 500 bp and 4 kbp, and λ-phage dsDNA molecules (˜48 kbp) can beused. DNA samples can be delivered from one chamber through the channel,using a common voltage polarity for each pore to promote capture fromone chamber and passage through the channel into the other chamber. Thelarge persistence length of dsDNA (one Kuhn length is 100 nm) ensuresthat the DNA segment inside each pore is likely fully extended androd-like. Voltage and ionic concentration can be varied to identifyadequate capture rates. Different buffered ionic concentrations can alsobe used in each chamber to enhance or alter capture rates, andconductance shift values that register the presence of DNA in each pore.

Using nanopore diameters 10 nm and larger minimizes the interaction (forexample, friction and sticking) between dsDNA and the nanopore walls.For larger pores, although dsDNA can be captured in an unfolded andfolded configurations, the single-file (unfolded) configuration is morelikely at higher voltages, and with shorter (≤3 kbp) dsDNA.

For an inter-pore distance of 500 nm or less, it is contemplated thatthe probability of dual-pore capture, following capture at the firstpore (either inlet or outlet) is very high, for voltages of at least 200mV in 1 M KCl (FIG. 3).

The radial distance within which voltage influence dominates thermaldiffusion, and leads to capture with high likelihood, has been estimatedto be at least 900 nm (larger than the inter-pore distance) for a rangeof pore sizes (6-15 nm diameter), voltages (120-500 mV), and with dsDNAat least 4 kbp in length (Gershow and Golovchenko, NatureNanotechnology, 2:775-779, 2007). These findings support a highlikelihood of prompt dual-pore capture of dsDNA, following single(first) pore capture of the dsDNA.

The capture and control of DNA through the two pores can benefit fromactive control hardware and real-time algorithms. The inventors havedeveloped active control hardware/software for DNA control. See, forexample, Gyarfas et al, Biophys. J., 100:1509-16, 2011); Wilson et al.,ACS Nano., 3(4):995-1003, 2009; and Benner et al., Nat. Nanotech.,2(11):718-24, 2007. A useful software is the LabVIEW software (Version8, National Instruments, Austin, Tex.), implemented on an FPGA(field-programmable gate array) system (PCI-7831R, NationalInstruments)). The referenced FPGA can control up to 4 amplifierssimultaneously. Further, the Axon Digidata 1440A Data Acquisition Systemused to digitize and log data onto a PC has 16 input channels, enough torecord voltage and current for up to 8 amplifiers in parallel. Otherreal-time operating system in concert with hardware/software forreal-time control and measurement could also be used for controlling theamplifiers, and digitizing and logging the data.

The inventors have also developed a low-noise voltage-clamp amplifiertermed the “Nanoclamp”, (Kim et al., IEEE Trans. On Biom. Circ. AndSyst. In press, May 2012; Kim et al., Elec. Lette., 47(15):844-6, July,2011; and Kim et al., Proceedings of the IEEE International SoC DesignConference (ISOCC), November, 2010) to functionalize and optimize theuse of one or more nanopores in small-footprint and multi-channeldevices. Any other commercial bench-top voltage-clamp or patch-clampamplifier, or integrated voltage-clamp or patch-clamp amplifier could beused for two pore control and measurement.

For a variety of solid-state pore materials and diameters, 0.1-10 kbptakes ˜1 ms to translocate. With a FPGA-controlled amplifier, one candetect capture and initiate competing voltage control within 0.020 ms,much faster than the 1 ms total passage time of 1 kbp DNA; thus,triggering the control method before DNA escapes (with no beadattachment) also has high likelihood. As demonstration of control, thetime to, and direction of, exit of the molecule from the pores can bedemonstrated as a function of the magnitude of and difference betweenthe competing voltages (FIG. 3). In single pore experiments, largefluctuations in the velocity of long WI kbp) dsDNA through the pore areexperimentally observed, and these fluctuations are too large to beattributed to diffusional Brownian motion. In (Lu, et al., BiophysicalJournal, 101(1):70-79, 2011), the dominant source of the net velocityfluctuations (that is, DNA length divided by total passage time) wasmodeled as being due to viscous drag induced by the voltage affectingportions of the DNA not yet in the pore, in addition to portion in thepore, where the voltage region of attraction extends. The model matchedexperimental data reasonably well. Notably, if the center of mass of thedsDNA is colinear with the pore upon capture, net velocity is faster,but if it is offset from the pore, net velocity is slower. Whencompeting voltages are engaged in the two-pore device, dsDNA velocitieswill not be affected by this viscous-drag-induced perturbation, unlessthe voltage difference is sufficiently high. The reason is that, afterdsDNA capture through both pores and competing voltage is engaged, thedsDNA between the pores will be fully extended and rod-like, andtherefore cannot engage in creating structures near either interior poreentry. On the other hand, dsDNA structures on the exterior sides of thepores are constantly forced by each local pore voltage away from themiddle channel, and are thus less likely to confound the pore entrykinetics. Such structure may affect the controlled delivery kinetics,and calibration experiments can be used to quantify this.

Force uncertainty induced by random transverse DNA motion is likelyminimal. Additionally, the voltage force causes an electroosmotic flow(EOF) in the opposite direction of DNA motion, causing the DNA to moveslower than it would in the absence of the induced counterion flow.Since different radial positions of the molecule can give rise todifferent EOF fields in the nanopore, one issue is whether the effectivecharge density and therefore the net driving force vary enough duringfluctuations in DNA radial position to induce speed fluctuations. It isbelieved that the effective charge density of DNA in 1M KCl is stablefor a distance of 1 nm or more between the pore wall and the DNA.

Additionally, SiN nanopores have a negative surface charge thatintrinsically repels DNA. Thus, although the molecule will undergoradial position fluctuations, by using SiN pores with diameter greaterthan a few nanometers, it is likely that each constant voltage valuewill result in a constant effective force at each of the two pores, andthus a constant velocity in the direction of larger force when using twocompeting voltages in the two-pore setup. Treatment of other porematerial surfaces can produce comparable effects to that of SiN.

Velocity uncertainty induced by random translational DNA motion that iscaused by Brownian motion may be reduced by increasing the competingvoltages. Experiments can be carried out to determine whether suchreduction will occur. A single-nanopore study (Lu, et al., BiophysicalJournal, 101(1):70-79, 2011) supports that increasing the competingforces can reduce uncertainty caused by Brownian motion. The studyanalyzed the velocity fluctuations caused by Brownian motion, whichoccur on fast (nanosecond) time scales, and the sequencing errors thatresult from such fluctuations. Assuming a hypothetical and idealizedsingle-nucleotide sensor (noise-free detection at >22 MHz bandwidth),Brownian motion alone results in 75% read error. The relevant parameterfor predicting the error is k_(B)T/F*(0.34 nm), which is the ratio ofthermal energy to the work done to translocate the DNA the distance abetween nucleotides (0.34 nm). In the ratio, force F=Vλ, is the voltageV driving DNA with charge density λ (0.2 e⁻/bp for dsDNA). For thepresent control method, increasing the voltage 50× results in 5% readerror, with higher voltage further improving errors. With a single pore,however, since mean velocity v is F*d/(k_(B)T) with diffusion constantd, DNA speed also increases with F, placing even more unrealisticdemands on the sequencing bandwidth.

To maintain the 22 MHz bandwidth, a 50× increase in force with a singlenanopore would have to be paired with a 50× increase in solutionviscosity to maintain the same v. Practically, however, 22 MHz bandwidthis already much higher than any experimental nanopore platform hasdemonstrated, or promises to demonstrate, for single-nucleotidesequencing. Moreover, increasing viscosity can slow DNA only up to 10×(Fologea, et al., Nano Lett., 5(9):1734-7, 2005) with single nanopores.Using the two-pore platform, each voltage can be kept sufficiently high,and this may suppress fluctuations caused by Brownian motion, while thedifferential voltage that determines the net DNA speed can be adjustedto ensure control rates are within actual sequencing bandwidths(nominally 1 kHz). An alternative method of suppressing Brownian motioninduced velocity fluctuations is to use feedback control. In one aspect,with 10 kHz bandwidth of the second pore current feedback to actuationof the first pore voltage at 10 kHz bandwidth, Brownian motion can becompensated to control detectable features on the DNA to remain in andnear the second pore at these kHz closed-loop bandwidths. Thiscapability is a one-dimensional analogue to the anti-Brownianelectrokinetic (ABEL) trap that suppressed Brownian motion in twospatial dimensions and works by optical forcing of beads attached tomolecules at Hz closed-loop bandwidths (Wang and Moerner, ACS Nano,5:5792-9, 2011). There is precedent for creating stiction force betweenDNA and (positively charged) nanopore walls. For instance, Bashir andcoworkers (Venkatesan et al., Adv. Mater., 20(8):1266-75, 2010;Venkatesan et al., Adv. Mater., 21:2771-6, 2009) reported the potentialfor Al₂O₃ pores to significantly reduce translocation rates due to theirunique ability to form positively charged crystal domains fromsputtering—the negatively charged phosphate backbone of DNA, whiletraversing an Al₂O₃ pore, interacts with the positive surface, slowingits movement. Recent work by Dekker and coworkers (Kowalczyk et al.,Nano. Lett., 12:1038-44, 2012) showed that LiCl slowed DNA rates by 10×,compared to the commonly used KCl ionic solution, provided a method thatany nanopore setup could benefit from.

Example 2. Detection and Localization of RecA Filaments Bound to DNA

This example shows that the two-chamber dual-pore device can be used tomap the binding of a DNA-binding protein to dsDNA, and for proteins thatdo or do not bind to specific sequences.

As demonstrated in Example 1, DNA samples can be captured from onechamber. RecA protein catalyses an ATP-dependent DNA strand-exchangereaction that pairs broken DNA with complementary regions of undamagedDNA. Using a poorly hydrolyzable ATP analogue ATP γS, RecA filamentsbound to dsDNA are very stable in high salt (for example, 1M KCl) whenfirst assembled in physiological salt. As an alternative to ATPγS, whichis slowly hydrolyzed, this example can also use ADP-AlF4 (aluminumtetrafluoride), which does not turnover at all, and causes RecA to bindmore tightly to the DNA.

Detection of RecA filaments bound to λ-DNA through 20-30 nm nanoporeshas been demonstrated (Kowalczyk et al., Nano Lett., 10(1):324-8, 2010;Smeets et al., Nano Lett., 9(9):3089-95, 2009; and Hall et al. NanoLett., 9(12):4441-5, 2009], but filaments <20 bp (6 or fewer RecAproteins) in length cannot be resolved using a single nanopore, due tothe coupling between translocation rate and measurement SNR.

Initial experiments of this example use bead-bound and unbound λ-DNAthat has been exposed to varying concentrations of RecA, to generate DNAthat is nearly uncoated, partially coated, and fully coated. Real-timemonitoring of each pore current can be used to gauge progress of thecontrolled delivery, and will be correlated for location mapping of thefilaments. Repeated measurements of each DNA will improve accuracy ofRecA mapping.

The added charge and bulk, and stability in high salt, when RecA isbound to DNA make it an ideal candidate to attempt detection andlocation mapping during controlled delivery with the proposedinstrument.

Control of RecA-bound DNA can also be attempted without a bead attachedto arrest translocation. As with dsDNA experiments in Example 1, activevoltage control can be used to promptly initiate competing voltagecontrol before the DNA exits the nanopores. As charged species that bindto DNA affect the mobility of DNA in an electric field, by altering thenet charge and stiffness of the DNA, motion control tuning experimentscan examine the influence of RecA binding to dsDNA on the force balanceused to control the motion of the dsDNA.

This example can demonstrate that the shortest observed filamentlengths, at low RecA concentrations, can be measured at high SNR and atsufficiently slow and controlled rates, so that any RecA protein boundin isolation can be detected if present.

The two-pore device therefore provides a completely new single-moleculeinstrument for basic research, as one could examine the capability todetect binding of additional proteins to the RecA-DNA filament, whichwould increase the filament width and thus be detected by a decrease inobserved current. For example, proteins that bind to the RecA-DNAfilament include LexA and bacteriophage lambda repressors, which useRecA to sense the status of the cell and switch on or off downstreamregulatory events.

Calibration experiments would involve detecting proteins that bind tospecific sequences (locations) on the DNA, so that protein-inducedshifts in the current would then permit estimation of the speed and ratecontrol performance of the DNA through the pores. Example proteins thatbind to specific sites on dsDNA include Lac repressor (binds to a 21 bpsegment), phage lambda repressor (which has multiple operator sites onλ-DNA), and other proteins.

Example 3. Detection and Localization of a Double-Stranded Segmentwithin a Singled-Stranded DNA

This example demonstrates the production of up to 10 kb ssDNA withdoubled-stranded segments of varying lengths.

In a first step, 10 kbp dsDNA can be created by long range PCR. One endof the strand is biotinylated for bead attachment, and the strands areseparated by chemical denaturing. The unbeaded 10 kb ssDNA then servesas the measured strand in two-chamber dual-pore experiments.Complementary single-stranded segments with desired sizes can be createdby PCR followed by bead capturing and strand separation.

ssDNA segments of varying lengths and at multiple sites within themeasured 10 kb ssDNA can be used, starting with a set of 100 ntsegments. Ionic current through a single solid-state pore was used todifferentiate dsDNA from ssDNA homopolymers, and purine and pyrimidinehomopolymers in (Skinner et al., Nano Lett., 9(8):2953-60, January2009). Thus, likelihood of differentiating single from double strandedsegments in DNA is high at sufficiently high voltage using the two-poredevice. Mapping ssDNA vs. dsDNA segments enables nanopore sequencingusing the hybridization-assisted method (though this method as proposedrelies on a costly hybridization-assisted process), and can be usedreveal both location and identity of target DNA sequences over longdistances (targeted sequencing). One can also explore the use of SingleStrand DNA Binding (SSB) proteins, as beads that will further amplifythe ssDNA vs. dsDNA differences in ionic current by binding to the ssDNAand creating a larger impedance than dsDNA.

Example 4. Capture and Control of Long ssDNA and Localization of RecA

This example demonstrates the capture and control of a long ssDNA andthe detection and localization of a RecA filament bound to the ssDNA.Additionally, it shows that the two-pore device can detect purine vs.pyrimidine homopolymeric segments within the ssDNA.

Stochastic detangling of 7 kb ssDNA through a 10 nm pore in a 20 nm SiNmembrane can be carried out as shown in Stefan et al., Nano Lett.,10:1414-20, 2010. While the single-nanopore method in Stefan et al. 2010unravels the ssDNA by the mechanical contact force between the tangledssDNA and the pore/membrane surface, it is contemplated that thedual-pore competing voltage setup can electrophoretically force ssDNA todetangle near and in between the pores at sufficiently high competingvoltages, by the action of each voltage force on the DNA backbonenearest each pore.

Detangling and subsequently precision control of the rate of ssDNAthrough the two pore setup is important for eventual sequencing of longssDNA molecules. At sufficiently high voltage (˜400 mV), it is possibleto discriminate purine and pyrimidine homopolymeric segments withinss-DNA (Skinner et al., Nano Lett., 9(8):2953-60, January 2009), whichis valuable for diagnostic applications and possibly cancer research.

This example also explores the use of RecA, or perhaps other SingleStrand DNA Binding (SSB) proteins, as detectable “speed-bumps” that aredifferentiable from the ssDNA ionic current by binding to the ssDNA andcreating a larger impedance. These speed bumps will allow directquantification of the controlled ssDNA speeds that are possible, whichin turn will demonstrate that the required 1 ms/nt is achievable. SinceRecA is not required to bind to specific trinucleotide sequence sites,but binds preferentially to TGG-repeating sequences and also tends tobind where RecA filaments are already formed, calibration experimentswill require the use of other ssDNA binding molecules that do bind tospecific known sequence locations. Having known binding sites that aredetectable as they pass through each pore is required to determine thespeed of the molecule as a function of the competing voltage values. Anon-limiting example is to use duplex strands (or bead-tethered duplexstrands) that hybridize to one or more known sites, from which theshifts in current could be used to detect passage of each duplex througheach pore, and then estimate the passing strand speed for the chosenvoltage values. Subsequently, RecA filaments can be formed and detectedon such molecules, keeping the duplex feature(s) as benchmark detectionpoints relative to which RecA filaments can be detected and theirposition inferred.

Methods for determining genetic haplotypes and DNA mapping byincorporating fluorescent labels into dsDNA (Xiao, et al., U.S. Pat. No.7,771,944 B2, 2010) can also use the two pore device, since the beadlabels (for example, quantum dots, or any fluorescent label) is bulkierand will produce shifts in the current just as binding proteins on dsDNAwould. Moreover, the two-pore method is simpler and much less expensivethan using high resolution imaging methods (that is, total internalreflection fluorescence microscopy) to detect and map the labelpositions. It is also noted that any velocity fluctuations caused byBrownian motion during controlled delivery are much less deleterious fordetecting larger features (proteins, duplex segments, bead attachments)than for detecting smaller features.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1.-20. (canceled)
 21. A system comprising: a channel through and definedby a plurality of layers surrounding the channel, the channel connectinga first chamber and a second chamber separated by the plurality oflayers, the plurality of layers comprising: a first layer, and aconductive layer coupled to the first layer and surrounding the channel,the channel comprising an inlet and an outlet defined by the pluralityof layers, the inlet and the outlet being between 10 nm and 1000 nmapart from each other, the conductive layer coupled to a power supply,wherein the system further comprises at least one sensor capable ofdetecting a polymer component during movement of the polymer componentinto the inlet, and wherein the sensor measures an ionic current throughthe inlet.
 22. The system of claim 21, wherein at least one of the inletand the outlet defines an opening that is from 0.5 nm to 100 nm indiameter.
 23. The system of claim 21, wherein at least one of the inletand the outlet has a depth from 0.1 nm to 100 nm.
 24. The system ofclaim 21, further comprising a first electrode coupled to the conductivelayer.
 25. The system of claim 24, further comprising a second electrodepositioned within the first chamber.
 26. The system of claim 21, whereinthe conductive layer is composed of at least one of gold, platinum,aluminum, and chrome.
 27. The system of claim 21, wherein the powersupply is configured to provide a first voltage V₁ between the firstchamber and the conductive layer, and a second voltage V₂ between theconductive layer and the second chamber.
 28. The system of claim 27,wherein the first voltage V₁ and the second voltage V₂ are independentlyadjustable.
 29. The system of claim 21, wherein the sensor is configuredto identify the polymer component by measuring one or more of: acurrent, a voltage, pH, an optical feature, and residence timeassociated with the polymer component.
 30. The system of claim 21,wherein the second chamber is coupled to a fluid channel in fluidcommunication with a reservoir of the system.
 31. The system of claim21, wherein the inlet is coaxial with the outlet.
 32. The system ofclaim 21, wherein the polymer component comprises a nucleotide, andwherein the system is configured to translocate the nucleotide from theinlet to the outlet with application of a voltage differential acrossthe first chamber and the second chamber by the power supply.
 33. Thesystem of claim 21, wherein the polymer component comprises a doublestranded DNA component.
 34. The system of claim 21, wherein the polymercomponent comprises a single stranded nucleic acid component.
 35. Amethod for controlling movement of a charged polymer through a channel,the method comprising: receiving a sample comprising a charged polymerinto one of a first chamber and a second chamber of a system, the systemcomprising: a channel through and defined by a plurality of layerssurrounding the channel, the channel fluidly coupling the first chamberand the second chamber, the plurality of layers comprising: a firstlayer, and a conductive layer coupled to the first layer, wherein thechannel comprises an inlet and an outlet defined by the plurality oflayers, and wherein the inlet and the outlet are between 10 nm and 1000nm apart from each other; and applying a differential voltage across thefirst chamber and the second chamber, thereby driving the chargedpolymer into the channel.
 36. The method of claim 35, wherein applyingthe differential voltage comprises setting a first voltage V₁ at thefirst chamber and a second voltage V₂ at the second chamber, therebytranslocating the charged polymer across the channel.
 37. The method ofclaim 36, further comprising adjusting the first voltage V₁ and thesecond voltage V₂ such that the first voltage V₁ and the second voltageV₂ generate forces to pull the charged polymer away from the conductivelayer.
 38. The method of claim 36, wherein the first voltage V₁ isdifferent in magnitude from the second voltage V₂, thereby controllablydriving the charged polymer through the channel in one direction. 39.The method of claim 38, wherein the first voltage and second voltage are10 to 10,000 times greater in magnitude than the difference between thefirst voltage V₁ and the second voltage V₂.
 40. The method of claim 35,wherein the charged polymer comprises one of: a double stranded DNAcomponent and a single stranded nucleic acid component.